Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
  • C12N15/877—Techniques for producing new mammalian cloned embryos
  • C12N15/8778—Swine embryos

Definitions

• the present invention in general, relates to a method of producing a cloned pig with a specific genetic character by gene targeting through introduction of a desired gene into somatic cells and somatic cell nuclear transfer, and pigs produced by such a method.
• the present invention relates to a cloned pig containing a specific gene, that is, green fluorescent protein (GFP) gene that encodes a protein emitting green color at a specific wavelength of light, and a method of producing such a pig.
• GFP green fluorescent protein
• the present invention is concerned with a cloned pig in which a gene responsible for the hyperacute rejection of xenografts from pigs, that is, alpha- 1,3-galactosyltransferase (GT) gene, is knocked out, and a method of producing such a pig.
• GT alpha- 1,3-galactosyltransferase
• the present invention relates to a gene targeting method comprising effectively introducing a GFP gene or a genetically manipulated GT gene into a cell. Further, the present invention relates to a vector capable of effectively removing a GFP gene or a genetically manipulated GT gene into a cell.
• the present invention indicates potential large-scale production of an animal disease model through successful introduction of a heterologous GFP gene into a pig, and makes it possible to produce a GT gene knock-out pig, thereby allowing pig organs to be transplanted into a human without hyperacute xenograft rejection.
• Transgenic animal technology has been under the spotlight for the past 20 years.
• the transgenic techniques are overwhelmingly important in terms of being capable of producing highly valuable products, and are widely used in biomedical and biological research.
• the transgenic techniques can be industrially applied in a broad range of applications from production of high-quality livestock products, high value-added pharmaceutically-active substances, animals having improved resistance to various pathogens and animal disease models, to genetic therapy.
• pronuclear microi ⁇ jection As a technique of introducing a heterologous gene into a cell to produce transgenic animals, pronuclear microi ⁇ jection, which was suggested by Gordon et al., is characterized by direct injection of a heterologous gene into a pronucleus of a fertilized oocyte, and widely applied to experimental animals including mice.
• pronuclear microi ⁇ jection method there are significant disadvantages with the pronuclear microi ⁇ jection method, as follows. When pronuclear microinjection is applied to industrial animals, production yield of transgenic animals is very low (0.5% in bovine, 1.5% in pigs, and 2.5% in sheep). In addition, genetic mosaicism occurs in most cases.
• transgenic animal cloning technique employs somatic cells transfected with a heterologous gene.
• the transgenic animal cloning technique can effectively produce transgenic cloned animals by generating reconstructed fertilized embryos with 100% transfection efficiency and without genetic mosaicism through nuclear transfer of only somatic cells transfected with a heterologous gene, and then transplanting the reconstructed embryos into surrogate mothers.
• sex of the transgenic animals can be artificially determined by analyzing in advance sex chromosomes of the transfected somatic cells, thereby maximizing their industrial usefulness.
• a desired gene When intended to produce transgenic pigs by somatic cell nuclear transfer, preferentially, a desired gene should be isolated and a vector carrying the desired gene should be constructed, and a molecular biological technique for introduction of the desired gene into somatic cells should be used along with a somatic cell cloning technique.
• the gene is typically isolated from a pig genomic DNA library by screening.
• the vector may be prepared according to intended use with consideration of an exogenous promoter, size of a gene of interest, positive or negative selectable markers, etc.
• the gene is introduced into nuclear donor cells by transfection using a biochemical method, a physical method, or virus-mediated gene transfer.
• biochemical method examples include calcium precipitation using calcium ions as a vehicle, lipofection using a cationic lipid that is a plasma membrane component, and a method using a non-lipid cationic polymer.
• transfection methods have been widely used owing to their simplicity, effectiveness and stability.
• the physical method includes electroporation, gene gun and intracytoplasmic microinjection.
• the virus-mediated gene transfer can be achieved by cloning a desired DNA into viral genome of adenovirus or retrovirus and then infecting cells with the resulting virus.
• the somatic cell cloning technique is disclosed in International Pat. Application No.
• PCT/KROO/00707 filed on June 30, 2000 by the present applicant, entitled “Method for Producing Cloned Cows", where somatic cell cloning is achieved by removing a nucleus containing genetic material from a cow oocyte and then injecting a nucleus from a different cell into the enucleated unfertilized oocyte.
• the resulting fertilized embryo is called "reconstructed embryo”. After being post-activated and cultured in vitro, the reconstructed embryo is transferred into a surrogate mother to produce live offspring.
• Organ transplantation in humans is a useful tool for treating organ-related incurable diseases, and has gradually increased for the past over 10 years. Relative to such increase of organ transplantation procedures, however, for the same period, the number of patients wanting to receive organ transplantation has increased three times.
• organ supply sources are seriously deficient, there is still no satisfactory method capable of solving the problem.
• Efforts to overcome such lack of organs for surgical transplantation in humans have been tried, which include development of artificial organs by medical engineering approaches and production of transgenic animals.
• pigs are typically selected as organ donors because of having similarity to humans in terms of physiological properties, size of the blood vessel system, and even diameter of erythrocytes.
• the use of pig organs is not problematic ethically, in comparison with primates.
• alpha-l,3-galactosyltransferase (GT) gene responsible for the formation of the xenoantigen, is disrupted in advance by gene targeting, thereby making it possible for xenografts from the resulting transgenic pig to be successfully transplanted into humans without hyperacute immune rejection of the xenografts, as well as not impairing the protective immune response in humans.
• GT alpha-l,3-galactosyltransferase
• the present invention provides methods of producing a cloned pig expressing green fluorescent protein (GFP) and an alpha- 1,3- galactosyltransferase (GT) gene knock-out cloned pig, and pigs produced by such methods, by gene targeting using a transfection method and somatic cell nuclear transfer.
• GFP green fluorescent protein
• GT alpha- 1,3- galactosyltransferase
• Fig. 1 is a photograph of a somatic cell expressing GFP
• Fig. 2 is a photograph of a nuclear transfer (NT) embryo obtained by fransferring a somatic cell expressing GFP into a recipient oocyte
• Fig. 3 is a photograph of a GFP-expressing nuclear transfer (NT) embryo at the blastocyst stage;
• Fig. 4 is a photograph showing transplantation of a transgenic nuclear transfer embryo into the oviduct of a surrogate mother
• Fig. 5 is a photograph showing a screening result of the primary pig genomic BAC library pool for GT gene
• Fig. 6 is a photograph showing a result of screening the secondary pig genomic
• Fig. 7 is a photograph showing a result of screening the tertiary pig genomic BAC library pool for GT gene
• Fig. 8 is a photograph showing a result of restriction mapping of a cloned GT gene.
• Fig. 9 is a schematic view of a vector for targeting of GT gene.
• the present invention is characterized by providing a transgenic cloned pig expressing a desired gene or having another desired gene knocked out, where the cloned pig is produced by gene targeting using a transfection method and somatic cell nuclear transfer.
• the present invention provides a transgenic cloned pig expressing GFP or having a GT gene knocked out by generating GPF-expressing or GT gene knock-out somatic cells using a transfection method, yielding reconstructed embryos by nuclear transfer, and transferring the reconstructed embryos into a surrogate mother.
• a method of producing a cloned pig expressing a GFP gene comprises the steps of (a) preparing a nuclear donor cell by culturing a cell line collected from a pig; (b) mixing a DNA construct carrying a GFP gene and a lipid component or non-lipid cationic polymer vehicle to form lipid (or cationic polymer)-DNA complexes, and adding the resulting complexes to a culture medium of the nuclear donor cell and further culturing the nuclear donor cell to introduce the GFP gene and express the GFP gene therein; (c) transferring the transfected nuclear donor cell into an enucleated pig recipient oocyte to generate a transgenic nuclear transfer (NT) embryo, and activating the NT embryo; and (d) transplanting the NT embryo into a surrogate mother pig to produce live offspring.
• NT transgenic nuclear transfer
• Step 1 Preparation, in vitro cultnring and maintenance of nuclear donor cells
• nuclear donor cells are needed.
• Several kinds of cells including somatic cell-derived cells and fertilized embryo-derived cells, are used as nuclear donor cells supplying nuclei in a nuclear transfer procedure.
• somatic fibroblasts isolated from pig fetuses are typically used.
• the fibroblasts have advantages in that a plurality of cells can be obtained at the initial step of fibroblast cell isolation, and they are relatively easy to culture and manipulate in vitro.
• fetal fibroblasts mainly used as nuclear donors
• crown-rump length of fetuses obtained from pregnant sows is measured, and length of gestation of the sows is calculated with reference to its breeding history.
• the fetal pigs are isolated by removing the fetal membrane, and then cutting the umbilical cord near the fetuses. Then, the fetal pigs are washed several times with phosphate-buffered saline (PBS) containing antibiotics and bovine serum albumin (BSA). After surgically removing the four legs, head and viscera from the body of the fetus, the body is again washed with PBS.
• PBS phosphate-buffered saline
• BSA bovine serum albumin
• the isolated fetal fibroblasts are incubated at 38°C under 95% humidity and 5% CO 2 .
• the culture is 90-100% confluent, the cells are subcultured, and the surplus cells are cryo-preserved.
• Step 2 Gene targeting by introduction of GFP gene into somatic cells pEGFP-Nl vector (Clontech Laboratories Inc., Palo Alto, CA), which is commercially available, is used in targeting of GFP gene.
• the pEGFP-Nl vector expresses a modified form of wild-type GFP, where the modified GFP has a high expression level and emits bright fluorescence.
• the vector is introduced into somatic cells using a biochemical vehicle, such as FuGENE 6 (Roche Diagnosis Corp. IN, USA),
• LipofectAmine Plus (Life Technologies) or ExGen 500 (MBI Fermentas).
• the FuGENE 6 transfection reagent which is a multi-component lipid based reagent, is advantageous in terms of having high transfection efficiency in a variety of cell types and low cytotoxicity, functioning both in the presence or absence of serum, and being easy to optimize its complex formation with DNA at a minimum volume.
• LipofectAmine Plus which is a cationic lipid
• ExGen 500 which is a non-lipid cationic polymer
• Cells into which a GFP gene are to be introduced are grown under optimal conditions, and subcultured by treatment with trypsin-EDTA to dissociate attached cells into single cells.
• trypsin-EDTA trypsin-EDTA
• the subcultured cells are fed with a fresh culture medium, and the medium is again exchanged with a fresh medium 4 hrs before transfection.
• the GFP gene is introduced into the cultured cells.
• lipid and non-lipid biochemical vehicles are used for targeting of the GFP gene by introduction of the GFP gene into nuclear donor cells.
• the GFP gene was mixed with a lipid or non-lipid vehicle to form complexes, and the resulting complexes were introduced into nuclear donor cells.
• To effectively introduce the GFP gene into the cells several parameters including amount of GFP gene DNA, volume of the vehicle, cell density, transfection time and addition or no addition of serum are selected, and optimized, thereby maximizing introduction efficiency and expression level of the GFP gene.
• Step 3 Selection, proliferation and crvo-preservation of nuclear donor cells transfected with GFP gene
• nuclear donor cells After being transfected with the GFP gene, nuclear donor cells are cultured for 3-5 days until the culture is completely confluent, where the GFP gene is integrated into chromosomes of the cells. Then, the cells are trypsinized, and the resulting single cells are observed under a fluorescence microscope equipped with a UV filter to select only green colored cells. In addition, nuclear donor cells transfected with the GFP gene are selected by in vitro culturing in the presence of a specific antibiotic.
• the pEGFP-Nl vector, carrying a GFP gene contains a neomycin-resistant gene that is used as a positive selectable marker.
• the neomycin-resistant gene is introduced into the cells along with the GFP gene, and expresses a neomycin-resistant protein in the cells. Therefore, when the targeted cells are cultured in a culture medium containing neomycin, only cells transfected with the vector survive, and cells not transfected with the vector die due to action of neomycin, resulting in proliferation of only the transfected cells in culture dishes (Fig. 1).
• Such selection using antibiotics may be effectively achieved by determining an optimal treatment concentration of antibiotics.
• the targeted cells are selected through treatment with neomycin for 2-3 weeks, where neomycin is added to the culture medium at a concentration of 200-800 ⁇ g/ml at intervals of 4-5 days.
• Cell proliferation pattern varies according to cell types. However, because cells are generally proliferated from one cell, the targeted cells should be proliferated at least up to a level required at the next step.
• the selected cells are cultured in a normal culture medium, where suitable growth factors and apoptosis-suppressing agents are added to the medium to induce rapid proliferation and reduce unnecessary loss of cells by apoptosis.
• suitable growth factors and apoptosis-suppressing agents are added to the medium to induce rapid proliferation and reduce unnecessary loss of cells by apoptosis.
• an optimal condition for cell storage is established, and the proliferated cells are cryo-stored at each passage.
• Step 4 Production of a reconstructed embryo by somatic cell nuclear transfer
• recipient oocytes are prepared by in vitro maturation of immature oocytes, as follows. Pig ovary is collected mainly in a slaughterhouse, tested for abnormalities, and washed three times with a proper washing solution. Then, immature oocytes are matured in vitro by cultiiring in a culture medium for maturation of the immature oocytes, that is, bovine serum albumin-free
• NCSU23 medium North Carolina State University 23 (NCSU23-M), see Table 1), containing 10% porcine follicular fluid (PFF), gonadotropic hormones (GTH), pregnant mare serum gonadotropin (PMSG) (Intervet Folligon), human chorionic gonadotropin (hCG) (intervet Chorulon), and epidermal growth factor (EGF) of 10 ng/ml.
• PFF porcine follicular fluid
• GTH gonadotropic hormones
• PMSG pregnant mare serum gonadotropin
• hCG human chorionic gonadotropin
• EGF epidermal growth factor
• recipient oocytes For nuclear transfer, recipient oocytes, nuclear donor cells, and pipettetes for cutting, enucleation and injection are prepared.
• Culture media are prepared using NCSU23 (NSCU23-W, see Table 2) washing medium as a basal medium.
• NCSU23 N-(NSCU23-W, see Table 2) washing medium as a basal medium.
• Each of recipient oocytes is put into NCSU23-W medium supplemented with 0.1% hyaluronidase to remove cumulus cells surrounding oocytes.
• the completely denuded oocytes are washed with a microdrop of NCSU23-W medium.
• the denuded oocytes are fixed with a holding pipette, a portion of the zona pellucida at an upper part of the first polar body is cut using a sharp pipette to give a slit.
• a portion of cytoplasm including the first polar body is removed by squeezing through the slit to generate enucleated oocytes.
• the enucleated oocytes are washed with NCSU23-W medium, and placed in a microdrop of NCSU23-M medium until nuclear transfer.
• the prepared nuclear donor cells are transferred to the enucleated recipient oocytes by aspirating the donor cells using an injection pipette after positioning the slit made on the zona pellucida of the oocytes to a straight line to the holding pipette, and injecting each of the donor cells into the perivitelline space of each of the enucleated oocytes through the slit, resulting in production of nuclear transfer embryos (Fig. 2).
• the nuclear transfer embryos are subjected to electrofusion, in which the enucleated oocytes are electrically fused with the donor cells with a single DC pulse of 1.8 kV/cm for 30 ⁇ sec using a BTX Electro Cell Manipulator (ECM2001, BTX, USA).
• ECM2001, BTX, USA BTX Electro Cell Manipulator
• NCSU23-W medium NCSU23-W medium
• NCSU23-D see Table 3
• the NCSU23 medium is supplemented with 10% serum.
• the reconstructed embryos are evaluated for development to the blastocyst stage and GFP expression (Fig. 3).
• Step 5 Transplantation of reconstructed embryos to surrogate mother pigs and production of live offspring
• Surrogate mother pigs suitable for transplantation of the reconstructed embryos and capable of developing the reconstructed embryos to normal fetuses are selected.
• the best time for the transplantation is determined by monitoring the estrus cycles of the selected sows. Generally, it is suitable for fertilization to be performed about 30-40 hours after a sow shows behavioral signs of estrus. Therefore, based on a suitable fertilization period, a proper time for embryo transplantation is calculated with consideration of time required for in vitro development of the reconstructed embryos.
• the reconstructed embryos are transferred to a surrogate mother pig by injecting the reconstructed embryos 2 cm deep in the oviduct, close to the ovary, after opening the abdomen of the surrogate mother by laparectomy (Fig. 4). 4 weeks after embryo transplantation, the sow is evaluated for pregnancy by ultrasound. After that, the ultrasonic diagnosis is carried out every two weeks to monitor the pregnancy of the surrogate mother and growth state of fetuses.
• calving is induced by injecting a hormone preparation into the mother sow, or by surgical operation such as Caesarean section.
• the present inventors produced a reconstructed embryo expressing GFP by nuclear transfer of somatic fibroblast cells transfected with a GFP gene to enucleated recipient embryos, and in vitro culturing of the resulting nuclear transfer embryos for 7 days to allow their development to the blastocyst stage.
• the reconstructed embryo was designated "SNU-P1 [Porcine NT Embryo]", and deposited at an international depositary authority, KCTC (Korean Collection for Type Cultures; KRIBB, 52, Oun- dong, Yusong-ku, Taejon, Korea) on Dec. 27, 2001, under accession number KCTC 10145BP.
• the present inventors obtained normal cloned offspring by transferring the reconstructed embryo to surrogate mother pigs.
• a method of producing a GT gene-knockout cloned pig comprises the steps of (a) preparing a nuclear donor cell by culturing a somatic cell line collected from a pig; (b) isolating a GT gene clone from a pig genomic BAC library, and constructing a gene targeting vector using the isolated GT gene, wherein the vector carries a GT gene modified by substituting a portion of a wild-type GT gene with a gene encoding a selectable marker by homologous recombination to suppress expression of a normal GT protein; (c) mixing the vector with a lipid or non-lipid component to form lipid (or non- lipid)-DNA complexes, and adding the resulting complexes to a culture medium of the nuclear donor cell to allow gene targeting by introducing the recombinant GT gene into the nuclear donor cell; (d) transferring the nuclear donor cells transfected with the recombinant GT gene into an enucleated pig recipient
• Step 1 Preparation, in vitro culturing and maintenance of nuclear donor cells
• nuclear donor cells are needed.
• Nuclear donor cells are prepared according to the same method as in Step 1 of the method of producing a cloned pig expressing a GFP gene.
• a GT gene is isolated by screening a pig genomic BAC library comprising three pools in total (Human Genome Mapping Project Inc., Great Britain). Primers to be used for the screening are prepared using the known pig GT cDNA sequence (GeneBank Accession No.: AF221517). To test specificity of primers and PCR method using the primers, PCR is carried out using pig genomic DNA and the primers, giving a positive PCR result. Using the primers, the three pig genomic BAC library pools are screened by PCR, and a single clone is obtained by PCR in which an amplified DNA fragment has an expected size. Then, the obtained GT gene clone is verified by Southern blotting.
• Step 3 Construction of a gene targeting vector carrying a knocked out GT gene and introduction of the vector into nuclear donor cells
• a gene targeting vector is prepared using the obtained GT gene clone.
• a GT gene is disrupted by substituting a portion of a GT gene with a gene encoding a selectable marker through homologous recombination, thereby preventing production of a normal GT protein.
• the vector is constructed not to have exogenous promoters by a promoter trap method.
• the vector comprises a nucleic acid sequence corresponding to a part of intron 8, exon 9 and a part of intron 9 of a GT gene, and a nucleic acid sequence encoding a puromycin-resistant gene linked to a SV40 poly(A) sequence, wherein the puromycin-resistant gene substitutes a nucleic acid sequence corresponding to an Aval-Drain fragment of the exon 9.
• the puromycin-resistant gene linked to SV40 poly(A) is inserted to the exon 9 of the GT gene by homologous recombination, thereby disrupting the GT gene (Fig. 9).
• the gene targeting vector is introduced into nuclear donor cells using FuGENE 6 mentioned in the method of producing a cloned pig expressing GFP.
• the resulting nuclear donor cells are cultured in a culture medium containing puromycin for 1-2 weeks to select targeted somatic fibroblasts. Thereafter, the selected somatic fibroblasts are confirmed by a method common in the art, including Southern blotting and PCR.
• Step 4 Production of a reconstructed embryo by somatic cell nuclear transfer This step is carried out according to the same procedure in Step 4 of the method of producing a cloned pig expressing GFP.
• Step 5 Transplantation of the reconstructed embryos to surrogate mother pigs and production of live offspring
• This step is carried out according to the same procedure in Step 5 of the method of producing a cloned pig expressing GFP.
• the present inventors produced a reconstructed embryo having a knocked out GT gene, by nuclear transfer of somatic fibroblast cells transfected with a vector having a knocked out GT gene into enucleated recipient embryos.
• the reconstructed embryo is designated "SNU-P2 [Porcine NT Embryo]", and deposited at an international depositary authority, KCTC (Korean Collection for Type Cultures; KRIBB, 52, Oun- dong, Yusong-ku, Taejon, Korea) on Dec. 27, 2001, under accession number KCTC 10146BP.
• the present inventors obtained normal cloned offspring by transferring the reconstructed embryo "SNU-P2" to surrogate mother pigs.
• EXAMPLE 1 Preparation, in vitro culturing and maintenance of nuclear donor cells
• cryo-preserved cells When reaching 90-100% confluency, cells were subcultured, and the surplus was cryo-preserved.
• the cryo-preserved cells were used as nuclear donors in somatic cell nuclear transfer, and subculture was carried out in a culture medium containing growth factors and an apoptosis suppressor to stimulate growth of cells and suppress cell death.
• EXAMPLE 2 Screening of pig BAC genomic library for GT gene
• pig genomic DNA was primarily prepared as follows. After obtaining about 5 g of ovary from a 6 month-pregnant Landrace sow, the obtained ovary was finely cut and ground in a mortar containing liquid nitrogen to destroy tissues. The ground tissue was treated with proteinase K at a concentration of 11 mg/ml and subjected to phenol extraction, thus giving pig genomic DNA.
• the primary pool is composed of 17 vials alphabetically marked from A to R (excluding K)
• the secondary pool is composed of 96-well plates with each of 15 individual pools
• the tertiary pool consists of 384-well plates for each pool of the secondary pool.
• PCR primer set consisting of a sense primer and an antisense primer was prepared: pig GT5 (5'-GAT CAA GTC CGA GAA GAG GTG GCA A-3'); and pig GT3 (5'-TCC TGG AGG ATT CCC TTG AAG CAC T-3').
• the expected PCR product is 342 bp in size.
• a PCR mixture was composed of 1 unit of Taq DNA polymerase, 10 mM dNTPs, 200 mM Tris-Cl (pH 8.8), 100 mM KC1, 100 mM (NIL ⁇ SO ⁇ 1% TritonX-100, lmg/ml of BSA, 100 ng/ ⁇ l of the pig genomic DNA and 2 ⁇ l of the primer set (40 pmol/ ⁇ l of a sense primer and 40 pmol/ ⁇ l of an antisense primer) in a total volume of 20 ⁇ l.
• PCR conditions included denaturation at 95°C for 5 min, and 40 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 1 min 30 sec, followed by final extension at 72°C for 15 min.
• the resulting PCR reaction mixture was analyzed by electrophoresis on an agarose gel.
• a PCR product was identified to be 342 bp in size, and was used as a positive control in screening the pig BAC genomic library.
• PCR was carried out with the primer set of pig GT5 and pig GT3 under the same condition as the PCR using the pig genomic DNA.
• the secondary pool (F: 76 to 90 plates; and G: 91 to 105 plates, each consisting of 15 pools) corresponding to the F and G pools showing a positive signal in the primary pool were screened by PCR under the same condition as described above, resulting in production of an amplified product having the identical size to that of the PCR using the pig genomic DNA.
• a PCR product of 342 bp in size was found in 81 and 82 of the F pool, and 91 of the G pool (Fig. 6).
• the 88 pool showed the strongest signal.
• EXAMPLE 3 Construction of a vector carrying a knocked out GT gene
• a probe for southern hybridization was prepared as follows. A DNA fragment of 351 bp in size, which corresponds to a part of the pig GT gene, was obtained by PCR and purified by gel electro-elution after electrophoresis on a 8% PAGE gel, and then labeled with ⁇ - 32 P[dCTP] using a random primer labeling kit (Life Technologies, USA).
• BAC DNA containing pig GT gene l ⁇ l of cloned E. coli from the 8F of the tertiary pool identified in Example 2 was primarily inoculated in 3 ml LB broth (CM+), and incubated at 37°C with agitation of 300 rpm for 12 hrs. Then, the cultured E. coli was inoculated again in 500 ml LB broth (CM+), and incubated for 16 hrs under the same condition.
• BAC DNA from the large-scale cultured E. coli was purified using a large- construct kit (Qiagen, Germany). Thereafter, 5 ⁇ g of the obtained BAC DNA was digested with 10 units of ⁇ coRI, HindHI, BamHI and Not!
• the identified BAC DNA fragment was cloned to pUC 19, as follows.
• pUC 19 vector was digested with ⁇ coRI for lhr 30 min, purified by phenol/chloroform extraction, and stored at -20°C until use.
• the BAC DNA fragment was mixed with 100 ng of the pUC 19 vector digested with ⁇ coRI, lOx ligation buffer and 2 ⁇ l of T4 DNA ligase (10 units/ ⁇ l) in a microtube, followed by incubation of 16 hrs at 15-16°C to perform ligation.
• exon 9 was found not to have three restriction enzyme recognition sites for ⁇ coRI, Hindi ⁇ and Notl, having only a BamHI site.
• the cloned BAC DNA fragment containing pig GT gene was treated with each of ⁇ coRI, HindHI, BamHI and Notl, and separated on a 1% agarose gel, where DNA bands of various sizes were found (Fig. 8). The gel was subjected to Southern hybridization. As a result, the DNA fragments containing exon 9 of the pig GT gene except for the BamHI fragment were found to be present as a single band, and have a molecular weight of about 8 to 12 kb.
• the EcoRI fragment was about 8 kb in size, and contained exon 9 of the pig GT gene and a part of two introns adjacent to exon 9. Therefore, after cleaving the cloned pig BAC DNA with EcoRI, the resulting EcoRI fragment was subcloned. Thereafter, a vector for gene targeting was prepared using the subcloned EcoRI fragment, as follows. To increase selection efficiency of targeted cells, the vector for gene targeting was prepared using a promoter trap strategy. The subcloned pig GT gene (1 ⁇ g) and a plasmid containing a puro cassette (Clontech) were digested with Aval and Drain, and Hindi ⁇ and BamHI, respectively, at 37°C for over 2 hrs.
• the digested products were treated with Klenow fragment DNA polymerase and dNTP to form blunt ends, followed by purification using a DNA elution kit (Qiagen, Germany) after electrophoresis on a 1% agarose gel.
• the purified GT gene fragment was ligated to a puromycin-resistant gene-SV40 poly(A) fragment using T4 DNA ligase, thereby giving a gene targeting vector (Fig. 9).
• EXAMPLE 4 Gene targeting by introduction of GFP gene and disrupted GT gene into fetal fibroblasts
• Pig fetal fibroblasts for gene targeting were prepared as follows. When grown to complete confluency in 60-mm culture dishes, fetal fibroblasts were washed with phosphate-buffered saline once after eliminating the culture medium, treated with 0.25% trypsin-EDTA, resuspended in 2 ml of a culture medium containing 10% FCS, and plated in 35 mm culture dishes. Next day, when the culture was reached 50-90% confluency, transfection of the fibroblast cells with GFP gene was performed. When using FuGENE 6, 1 ⁇ g of a DNA sample and 3 ⁇ l of FuGENE 6 were introduced into each well of a 35-mm culture dish, containing fibroblasts.
• a serum-free culture medium was aliquotted into Eppendorf tubes. 1 ⁇ g of pEGFP-Nl vector DNA and 3 ⁇ l of FuGENE 6 were sequentially added to each tube, followed by pulse centrifugation for 10 sec at 3000 rpm. After being incubated at room temperature for 15 min, 100 ⁇ l of the mixture was added to each well of the 35 mm culture dish, and the dish was swirled and incubated in a CO incubator.
• the cationic liposome LipofectAmin plus (Life Technologies) has an advantage in terms of having high transfection efficiency even when using a small amount of DNA.
• Phosphate-buffered saline and an FCS/antibiotics-free culture medium were pre-warmed at 37°C 30 min before use.
• 100 ⁇ l of a serum-free culture medium or Opti-MEM and 4 ⁇ l of Plus reagent were mixed, and added to the tube.
• the mixture was incubated at room temperature for 15 min.
• a 6-well plate containing 90%-confluent fetal fibroblasts was washed twice with the phosphate-buffered saline.
• the DNA mixture was added to each well, and the plate was swirled, followed by incubation in a CO 2 incubator.
• the cationic polymer reagent ExGen 500 MMI Fermentas
• 2 ⁇ l of pEGFP-Nl vector DNA was mixed with 100 ⁇ l of 150 mM NaCl and then 6.6 ⁇ l of ExGen 500, and the DNA mixture was pulse- centrifuged at 3000 rpm for 10 sec. After being incubated for 10 min at room temperature, the DNA mixture was added to each well of a 35 mm culture dish containing fetal fibroblasts grown to 60% confluency, followed by incubation in a CO 2 incubator.
• EXAMPLE 5 Selection, proliferation and cryo-preservation of nuclear donor cells transfected with GFP gene
• the pig fetal fibroblasts transfected with GFP genes using three different transfection reagents were cultured for 3-5 days until reaching complete confluency, and detached and separated into single cells by trypsmization. The single cells were observed under a microscope equipped with a UV filter to identify cells expressing GFP protein.
• the cells was incubated a culture medium supplemented with neomycin for 3 weeks, in which neomycin was added to the medium at a concentration of 400 ⁇ g/ml at intervals of 4-5 days. After selection, formed colonies were trypsinized, and cultured in 96-well plates after suitable dilution. The proliferated cells in each well of the 96-well plates were transferred to 24-well plates, and further to 12-well and then 6-well plates, followed by incubation.
• genomic DNA was isolated from an established clone. Using the isolated genomic DNA, when performing PCR using a primer set designated as the following sequences: 5'- GCGATGCCACCTACGGCAAGCTGA-3' and 5'-
• the identified cloned pig fetal fibroblasts were cryo-preserved by suspending the proliferated cells in a freezing medium prepared using a 10% FCS-containing culture medium and 15% FCS, placing the suspended cells at 4°C for 2 hrs and then at -70°C for 12 hrs, and storing the frozen cells at -150°C.
• Follicles of about 3-6 mm in diameter were aspirated from pig ovary collected from a slaughterhouse using a 5 ml syringe with an 18-gauge needle. After transferring the follicles to a 100 mm dish having square lattice (lxl cm) lines, oocytes surrounded by sufficient cumulus cells and having homogeneous cytoplasm were selected. The selected oocytes were washed with 2 ml of NCSU23-W medium in a 35 mm culture dish three times, and finally washed with NCSU23-M medium.
• FCS-free NCSU23-M medium was supplemented with 10% porcine follicular fluid (PFF), GTH, PMSG, hCG and 10 ng/ml of EGF, and 480 ⁇ l of the medium was aliquotted into each well of 4-well plates. 50-60 immature oocytes were put into each well of the plates, and incubated for 22 hrs under 5% CO 2 . Then, the oocytes were matured in vitro in NCSU23-M medium not containing the hormones as described above for 20-22 hrs.
• PFF porcine follicular fluid
• EXAMPLE 7 Somatic cell nuclear transfer
• the recipient oocytes prepared in Example 4 were washed with NCSU23-W medium once, and transferred into NCSU23-W containing 0.1% hyaluronidase. Then, cumulus cells were eliminated from the recipient oocytes.
• the denuded oocytes were transferred into a cytochalasin B solution prepared by mixing 1 ⁇ l of cytochalasin B (Sigma Chemical Co., USA) dissolved in DMSO (dimethyl sulfoxide) at a concentration of 7.5 mg/ml with 1 ml of NCSU23-W medium supplemented with 10% FCS.
• DMSO dimethyl sulfoxide
• a holding pipette was rubbed with a sharp micropipette penetrating the zona pellucida of the oocytes to form a slit. Then, 10-15% of cytoplasm was removed from the oocytes by squeezing on their upper part with the sharp micropipette, resulting in production of enucleated oocytes.
• the nuclear donor cells prepared in advance were transferred into the enucleated recipient oocytes.
• a 4 ⁇ l injection microdroplet was placed on the middle of an upper part of a working dish using a PHA-P (phytohemagglutinin) solution prepared by mixing 100 ⁇ l of a PHA-P stock solution prepared by dissolving 5 mg of PHA-P in 10 ml of NCSU23-W medium with 400 ⁇ l of NCSU23-W medium.
• two microdroplets for nuclear donor cells were made above and below the injection microdroplet of the working dish using 4 ⁇ l of PBS containing 0.5% FCS. After covering the microdroplets with mineral oil, the working dish was placed on a micromanipulator plate.
• the enucleated oocytes in NCSU-M medium were washed with NCSU-W medium three times, and transferred into the injection microdroplet. Then, the nuclear donor cells were transferred into the injection microdroplet using an injection pipette. Using the injection pipette, cells identified to express GFP or cells having a GT gene knocked out were injected into the perivitelline space of the enucleated recipient oocytes through the slit (Fig. 3). The resulting transgenic nuclear transfer (NT) embryos were washed with NCSU-W medium three times, and placed into NCSU-W medium.
• EXAMPLE 8 Cell fusion and activation
• the transgenic NT embryos were subjected to electrofusion using a BTX Electro cell manipulator (BTX, USA), as follows. 15 ⁇ l of a mannitol solution (see Table 4) was added to the NCSU23-W medium containing the NT embryos using a mouth pipette for washing, followed by incubation for 1 min. The NT embryos were incubated for 1 min in a mannitol solution containing NCSU23-W medium, and suspended in the mannitol solution used for their washing, using the mouth pipette.
• BTX BTX Electro cell manipulator
• the NT embryos were placed in a chamber with electrodes at each end, containing a mannitol solution and connected to the BTX Electro cell manipulator, in an orientation in which the nuclear donor cells face to the cathode. Thereafter, cell fusion of the NT embryos was induced by applying once a DC pulse of 1.8 kV/cm for 30 ⁇ sec. Within 20 min after the electric stimulation, the NT embryos were viewed under a microscope to determine whether cell fusion was achieved, where unfused NT embryos were sujected to electrofusion again. The NT embryos identified to be fused were transferred into NCSU23-W medium, where the NT embryos were activated.
• the electrofused transgenic NT embryos were incubated in NCSU23-D medium. After 4 days of culturing, the NCSU23- D medium was supplemented with 10% FCS. On day 7, each of the transgenic NT embryos was evaluated for development to the blastocyst stage and GFP expression, where GFP expression was investigated under UV illumination (Fig. 4).
• EXAMPLE 10 Comparison of development levels of NT embryos according to use of nuclear donor cells transfected with GFP gene or not
• the nuclear donor cells transfected with a GFP gene, prepared in Example 4, and normal somatic fibroblast cells were subjected to somatic cell nuclear transfer according to the same method in Examples 6 to 9.
• division rates, development rates to the blastocyst stage and cell number in the blastocyst stage were analyzed (see Table 5).
• Table 5 it was found that there is no significant difference in development levels of the nuclear transfer embryos between the cases of introducing the GFP gene into the donor cells or not, indicating that the introduction of the GFP gene into fibroblast cells does not affect the development of nuclear transfer embryos.
• EXAMPLE 11 Comparison of development levels of NT embryos according to introduction methods of GFP gene into nuclear donor cells
• nuclear donor cells were transfected with a GFP gene using each of the three transfection reagents used in Example 4, and somatic cell nuclear transfer was performed according to the same method as in Examples 6 to 9.
• EXAMPLE 12 Transplantation of NT embryos into surrogate mothers
• the selected nuclear transfer embryos were injected 2 cm-deep of the oviduct, close to the ovary (Fig. 5), together with phosphate-buffered saline containing 20%) FCS.
• the surrogate sows were anesthetized by being intramuscularly injected with the general anesthetic atropine at an amount of 1 mg/kg body weight and then with the tranquilizer azaperrone (Stresnil, P/M; Mallinckrodt) at an amount of 2-4 mg/kg, and, after 10 min, with ketamine HCl at an amount of 20 mg/kg.
• NT embryos were injected through the catheter. After confirming successful injection of the transgenic NT embryos using a microscope, 500 ml of a physiological saline solution containing antibiotics was injected into the inside of the abdomen. Then, the opened abdomen was sutured with biosorbent suture thread. After the surgery, a broad range of antibiotics was administered to the surrogate sows for 5 days to prevent infection.
• EXAMPLE 13 Evaluation of pregnancy of the surrogate sows and production of live offspring expressing GFP and carrying a knocked out GT gene
• the surrogate mothers were evaluated for pregnancy by an ultrasonic diagnostic system.
• the ultrasonic diagnosis was carried out every two weeks to monitor the pregnancy of the surrogate mothers. 114 days after the embryonic transplantation, 7 cloned piglets were born from the surrogate mothers expressing GFP, and 3 cloned piglets were bom from the GT gene knock-out surrogate mothers.
• EXAMPLE 14 Genetic analysis of transgenic cloned pigs
• Example 13 Genetic analysis of the live offspring produced in Example 13 was carried out by molecular biological methods, and their phenotype was evaluated with the naked eye.
• the live offspring were evaluated for GFP expression and introduction of the knocked out GT gene by the naked eye, as well as by performing Southern blotting, Western blotting and cell culture using their tissues.
• the offspring were evaluated for GFP expression by investigating induction of green color in their skin, mouths and tongues with the naked eye. Also, to investigate GFP expression in the offspring, genomic DNA from the offspring was analyzed by Southern blotting, and protein samples of some tissues were analyzed by Western blotting. As a result, the offspring were found to express GFP. In addition, when analyzing the live offspring bom from the surrogate mothers into which the embryos carrying a knocked out GT gene by Southern blotting, the offspring were found to have a knocked out GT gene.
• the present invention provides a cloned pig expressing GFP and a cloned pig carrying a GT gene knocked out by transfecting somatic cells with a GFP gene or a disrupted GT gene, and nuclear transfer of the resulting somatic cells into recipient oocytes, thereby making it possible to produce an animal disease model in a large-scale, as well as an animal able to supply organs transplantable into humans without hyperacute immune rejection.

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